The ontogeny of the human globin genes is an important focus of study both with respect to the fundamental developmental biology and molecular genetics of the control of expression of this complex gene system, but also because modifying this developmental control would be of therapeutic value in the treatment of the prevalent genetic diseases of hemoglobin. We are studying this problem from several aspects. First, we are preparing knock-out mice which lack segments of the control region 5' of the mouse epsilon globin gene (homologous to that in man) which we have previously shown have important regulatory functions -mostly as silencers - in the expression of this gene. We are now testing our Cre-Lox constructs for these knock-outs in ES cells. Second, we are studying the effects of GATA-1 and -2 expression on endogenous globin gene expression and are examining the relationship of chromatin acetylation to the expression of individual globin genes, especially the gamma genes. Third, we are working to improve culture methods for human erythroid precursors, by antibody selection, to obtain purified populations of these cells to establish at which developmental stage drugs change the phenotype. Fourth, we have developed real-time, quantitative PCR methods to measure globin gene expression in single cells and are using these methods to analyze the mechanisms of the effects of chemically useful drugs, such as hydroxyurea and butyrate, on globin gene expression. Fifth, we are using the cells to see if we can predict which SS patients will respond to HU treatment. Lastly, we are using variants of the DNA-chip technology to develop methods to diagnose the genetic diseases of hemoglobin and to study the effects of drugs on erythroid cell phenotype. These studies, in conjunction with our previous robotic drug-discovery efforts, have allowed us to begin to identify new agents - and to understand their mechanisms of action - which may be of use in treating sickle cell disease and the thalassemia syndromes.